The invention relates to a process for the microbial production of amino acids, to export genes, to regulator genes, to vectors, to transformed cells, to membrane proteins, and to uses.
Amino acids are of high economical interest and there are many applications for the amino acids: for example, L-lysine as well as L-threonine and L-tryptophan are needed as feed additives, L-glutamate as seasoning additive, L-isoleucine, and L-tryosine in the pharmaceutical industry, L-arginine and L-isoleucine as medicine or L-glutamate and L-phenylalanine as a starting substance for the synthesis of fine chemicals.
A preferred method for the manufacture of these different amino acids is the biotechnological manufacture by means of micro-organisms; since, in this way, the biologically effective and optically active form of the respective amino acid is directly obtained and simple and inexpensive raw materials can be used. As microorganisms, for example, Corynebacterium glutamicum and its relatives ssp. flavum and ssp lactofermentum (Liebl et al; Int. J-System Bacteriol (1991) 41:255-260) as well as Escherichia coli and related bacteria can be used.
However, these bacteria produce the amino acids only in the amounts needed for their growth such that no excess amino acids are generated and are available. The reason for this is that in the cell the biosynthesis of the amino acids is controlled in various ways. As a result, different methods of increasing the formation of products by overcoming the control mechanisms are already known. In these processes, for example, amino acid analogs are utilized to render the control of the biosynthesis ineffective. A method is described, for example, wherein Corynebacterium strains are used which are resistant to L-tyrosine and L-phenylalanine analogs (JP 19037/1976 and 39517/1978). Also methods have been described in which bacteria resistant to L-lysine and also to L-threonine analogs are used in order to overcome the control mechanisms (EP 0 205 849 B1, UK patent application GB 2 152 509 A).
Furthermore, microorganisms constructed by recombinant DNA techniques are known wherein the control of the biosynthesis has also been eliminated by cloning and expressing the genes which code for the key enzymes which cannot be feed-back inhibited any more. For example, a recombinant L-lysinee producing bacterium with plasmid-coded feedback-resistant aspartate kinase is known (EP 0381527). Also, a recombinant L-phenylalanine producing bacterium with feedback resistant prephenate dehyrodgenase has been described (JP 124375/1986; EP 0 488 424). In addition, increased amino acid yields have been obtained by overexpression of genes which do not code for feedback-sensitive enzymes of the amino acids synthesis. For example, the lysine formation is improved by increased synthesis of the dihydrodipicolinate synthase (EP 0 197 335). Also, the threonine formation is improved by increased synthesis of threonine dehydratase (EP 0 436 886 Al).
Further experiments for increasing the amino acid production aim at an improved generation of the cellular primary metabolites of the central metabolism. In this connection, it is known that the overexpression of the transketolase achieved by recombinant techniques improve the product generation of L-tryptophan, L-tyrosine or L-phenalanine (EP 0 600 463 A2). Furthermore, the reduction of the phosphenol pyruvate carboxylase activity in Corynebacterium provides for an improvement in the generation of aromatic amino acids (EP 0 331 145).
All these attempts to increase the productivity have the aim to overcome the limitation of the cytosolic synthesis of the amino acids. However, as a further limitation basically also the export of the amino acids formed in the interior of a cell into the culture medium should be taken into consideration. As a result, it has been tried to improve this export and, consequently, the efficiency of the amino acid production. For example, the cell permeability of the Corynebacterium has been increased by biotin deficiency, detergence or penicillin treatment. However, these treatments were effective exclusively in the production of glutamate, whereas the synthesis of other amino acids could not be improved in this manner. Also, bacteria strains have been developed in which the activity of the secretion system is increased by chemical or physical mutations. In this way, for example, a Corynebacterium glutamicum strain has been obtained which has an improved secretion activity and is therefore especially suitable for the L-Lysine production. (DE 02 03 320).
Altogether, the attempts to increase the secretion of amino-acids formed within the cell have all in common that an increase efflux of amino acids on the basis of the selected non-directed and non-specific methods could be achieved only accidentally.
Solely in the German patent application No. 195 23 279.8-41, a process is described which provides for a well-defined increase of the secretion of amino acids formed internally in a cell by increasing the expression of genes coding for the import of amino acids. The understanding on which this process was based, that is, the cell utilizes import proteins for the export of amino acids as well as the fact that by nature microorganisms do not generate and release excess amino acids lets one assume that export genes or proteins specific for the amino acid transport do not exist, but that the amino acids are excreted by way of other export systems.
The export systems known so far export poisonous metal ions, toxic antibiotica and higher molecular toxins. These export systems are relatively complex in their structure. Generally, membrane proteins of the cytoplasmic membrane are involved which however cause only a partial reaction of the export so that presumably additional extra cytoplasmic support proteins are needed for the transport (Dink, T. et al., A family of large molecules across the outer membranes of gram-negative bacteria., J. Bacteriol. 1994, 176: 3825-3831). Furthermore, it is known that, with the sec-dependent export system for extra-cellular proteins, at least six different protein components are essential for the export. This state-of-the-art suggests that also the systems, which are responsible for the export of amino acids, but which are not known so far comprise several protein components or respectively, several genes are responsible for the export of amino acids. A hint in this direction could be the various mutants which are defective in the lysine export as described by Vrylic et al.,(J. Bacteriol (1995) 177:4021-4027).